Transgenic Research

, Volume 17, Issue 5, pp 863–872 | Cite as

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland

  • Hernan BaldassarreEmail author
  • Duncan K. Hockley
  • Benjamen Olaniyan
  • Eric Brochu
  • Xin Zhao
  • Arif Mustafa
  • Vilceu Bordignon
Original Paper


The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1–5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na+, K+, assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K+ concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats.


Cholinesterase Transgenic Tight junction Fatty acid composition Caprine serum albumin Caseins 


  1. AOAC (1999) Official methods of analysis. Association of Official Analytical Chemists, Gaithersburg MD, USAGoogle Scholar
  2. Baldassarre H, Karatzas CN (2004) Advanced assisted reproduction technologies (ART) in goats. Anim Reprod Sci 82–83:255–266PubMedCrossRefGoogle Scholar
  3. Baldassarre H, Wang B, Keefer CL, Lazaris A, Karatzas CN (2004) State of the art in the production of transgenic goats. Reprod Fertil Dev 16:465–470PubMedCrossRefGoogle Scholar
  4. Baldassarre H, Hockley DK, Dore M, Brochu E, Hakier B, Zhao X, Bordignon V (2008) Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk. Transgenic Res 17:73–84PubMedCrossRefGoogle Scholar
  5. Bernard L, Rouel J, Leroux C, Ferlay A, Faulconnier Y, Legrand P, Chilliard Y (2005) Mammary lipid metabolism and milk fatty acid secretion in alpine goats fed vegetable lipids. J Dairy Sci 88:1478–1489PubMedGoogle Scholar
  6. Cook KK (1997) Extension of dry ash atomic absorption and spectrophotometric methods to determination of minerals and phosphorus in soy-based, whey-based, and enteral formulae (modification of AOAC Official Methods 985.35 and 986.24): collaborative study. J AOAC Int 80:834–844PubMedGoogle Scholar
  7. Cudna RE, Dickson AJ (2003) Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 81:56–65PubMedCrossRefGoogle Scholar
  8. de Groot N, van Kuik-Romeijn P, Lee SH, de Boer HA (2001) Over-expression of the murine pIgR gene in the mammary gland of transgenic mice influences the milk composition and reduces its nutritional value. Transgenic Res 10:285–291PubMedCrossRefGoogle Scholar
  9. Doctor BP, Saxena A (2005) Bioscavengers for the protection of humans against organophosphate toxicity. Chem Biol Interact 157–158:167–171PubMedCrossRefGoogle Scholar
  10. Echelard Y, Ziomek CA, Meade HM (2006) Production of recombinant therapeutic proteins in the milk of transgenic animals. Biopharm Int 19:36–46Google Scholar
  11. Ellman GC, Courtney KP, Andres V Jr, Featherstone RM (1961) A new rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95PubMedCrossRefGoogle Scholar
  12. Gerson T, Shorland FB, Wilson GF, Reid CWS (1968) Origin of glyceride fatty acids in cow milk fat. J Dairy Sci 51:356–361PubMedGoogle Scholar
  13. Guzman AA, Perez MD, Marquez RM, Garza JR (1986) Colorimetric determination of bovine serum albumin in milk using bromocresol green dye. J Dairy Sci 69(Suppl 1):102 AbstractGoogle Scholar
  14. Houdebine LM (2000) Transgenic animal bioreactors. Transgenic Res 9:305–320PubMedCrossRefGoogle Scholar
  15. Huang YJ, Huang Y, Baldassarre H, Wang B, Lazaris A, Leduc M, Bilodeau AS, Bellemare A, Cote M, Herskovits P, Touati M, Turcotte C, Valeanu L, Lemee N, Wilgus H, Begin I, Bhatia B, Rao K, Neveu N, Brochu E, Pierson J, Hockley DK, Cerasoli DM, Lenz DE, Karatzas CN, Langermann S (2007) Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning. Proc Natl Acad Sci USA 104:13603–13608PubMedCrossRefGoogle Scholar
  16. Jhappan C, Geiser AG, Kordon EC, Bagheri D, Hennighausen L, Roberts AB, Smith GH, Merlino G (1993) Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. EMBO J 12:1835–1845PubMedGoogle Scholar
  17. Keefer CL (2004) Production of bioproducts through the use of transgenic animal models. Anim Reprod Sci 82–83:5–12PubMedCrossRefGoogle Scholar
  18. Kelly ML, Kolver ES, Bauman DE, Van Amburgh ME, Muller LD (1998) Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows. J Dairy Sci 81:1630–1636PubMedGoogle Scholar
  19. Kondyli E, Katsiari MC, Voutsinas LP (2007) Variations of vitamin and mineral contents in raw goat milk of the indigenous Greek breed during lactation. Food Chem 100:226–230CrossRefGoogle Scholar
  20. Lieske B, Jantz A, Finke B (2005) An improved analytical approach for the determination of bovine serum albumin in milk. Lait 85:237–248CrossRefGoogle Scholar
  21. Linzell JL, Peaker M (1971) Mechanism of milk secretion. Physiol Rev 51:564–597PubMedGoogle Scholar
  22. Lockridge O, Bartels CF, Vaughan TA, Wong CK, Norton SE, Johnson LL (1987) Complete amino acid sequence of human serum cholinesterase. J Biol Chem 262:549–557PubMedGoogle Scholar
  23. Matsushita M, Tazinafo NM, Padre RG, Oliveira CC, Souza NE, Visentainer JV, Macedo FAF, Ribas NP (2007) Fatty acid profile of milk from Saanen goats fed a diet enriched with three vegetable oils. Small Rumin Res 72:127–132CrossRefGoogle Scholar
  24. McClenaghan M, Springbett A, Wallace RM, Wilde CJ, Clark AJ (1995) Secretory proteins compete for production in the mammary gland of transgenic mice. Biochem J 310(Pt 2):637–641PubMedGoogle Scholar
  25. Nguyen DA, Neville MC (1998) Tight junction regulation in the mammary gland. J Mammary Gland Biol Neoplasia 3:233–246PubMedCrossRefGoogle Scholar
  26. Ollier S, Robert-Granie C, Bernard L, Chilliard Y, Leroux C (2007) Mammary transcriptome analysis of food-deprived lactating goats highlights genes involved in milk secretion and programmed cell death. J Nutr 137:560–567PubMedGoogle Scholar
  27. Palmer CA, Lubon H, McManaman JL (2003) Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development. Transgenic Res 12:283–292PubMedCrossRefGoogle Scholar
  28. Park YW, Juarez M, Ramos M, Haenlein GFW (2007) Physico-chemical characteristics of goat and sheep milk. Small Rumin Res 68:88–113CrossRefGoogle Scholar
  29. Reh WA, Maga EA, Collette NM, Moyer A, Conrad-Brink JS, Taylor SJ, DePeters EJ, Oppenheim S, Rowe JD, Bondurant RH, Anderson GB, Murray JD (2004) Hot topic: using a stearoyl-CoA desaturase transgene to alter milk fatty acid composition. J Dairy Sci 87:3510–3514PubMedGoogle Scholar
  30. Schroder M (2006) The unfolded protein response. Mol Biotechnol 34:279–290PubMedCrossRefGoogle Scholar
  31. Scriven P, Brown NJ, Pockley AG, Wyld L (2007) The unfolded protein response and cancer: a brighter future unfolding? J Mol Med 85:331–341PubMedCrossRefGoogle Scholar
  32. Shennan DB, Peaker M (2000) Transport of milk constituents by the mammary gland. Physiol Rev 80:925–951PubMedGoogle Scholar
  33. Stelwagen K, Farr VC, Davis SR, Prosser CG (1995) EGTA-induced disruption of epithelial cell tight junctions in the lactating caprine mammary gland. Am J Physiol 269:R848–R855PubMedGoogle Scholar
  34. Stelwagen K, Farr VC, McFadden HA, Prosser CG, Davis SR (1997) Time course of milk accumulation-induced opening of mammary tight junctions, and blood clearance of milk components. Am J Physiol 273:R379–R386PubMedGoogle Scholar
  35. Stelwagen K, van Espen DC, Verkerk GA, McFadden HA, Farr VC (1998) Elevated plasma cortisol reduces permeability of mammary tight junctions in the lactating bovine mammary epithelium. J Endocrinol 159:173–178PubMedCrossRefGoogle Scholar
  36. Stelwagen K, Farr VC, McFadden HA (1999) Alteration of the sodium to potassium ratio in milk and the effect on milk secretion in goats. J Dairy Sci 82:52–59PubMedGoogle Scholar
  37. Ward AT, Wittenberg KM, Przybylski R (2002) Bovine milk fatty acid profiles produced by feeding diets containing solin, flax and canola. J Dairy Sci 85:1191–1196PubMedCrossRefGoogle Scholar
  38. Wheeler MB, Walters EM, Clark SG (2003) Transgenic animals in biomedicine and agriculture: outlook for the future. Anim Reprod Sci 79:265–289PubMedCrossRefGoogle Scholar
  39. Wilde CJ, Clark AJ, Kerr MA, Knight CH, McClenaghan M, Simons JP (1992) Mammary development and milk secretion in transgenic mice expressing the sheep beta-lactoglobulin gene. Biochem J 284(Pt 3):717–720PubMedGoogle Scholar
  40. Zhang RH, Mustafa AF, Ng-Kwai-Hang KF, Zhao X (2006) Effects of freezing on composition and fatty acid profiles of sheep milk and cheese. Small Rumin Res 64:203–210CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Hernan Baldassarre
    • 1
    • 2
    Email author
  • Duncan K. Hockley
    • 1
  • Benjamen Olaniyan
    • 2
  • Eric Brochu
    • 1
  • Xin Zhao
    • 2
  • Arif Mustafa
    • 2
  • Vilceu Bordignon
    • 2
  1. 1.Pharmathene Inc.St. TelesphoreCanada
  2. 2.Department of Animal ScienceMcGill UniversityMontrealCanada

Personalised recommendations